Fungal and Host Transcriptome Analysis of Ph-Regulated Genes

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Fungal and Host Transcriptome Analysis of Ph-Regulated Genes Barad et al. BMC Genomics (2016) 17:330 DOI 10.1186/s12864-016-2665-7 RESEARCH ARTICLE Open Access Fungal and host transcriptome analysis of pH-regulated genes during colonization of apple fruits by Penicillium expansum Shiri Barad1,2†,NoaSela3†, Dilip Kumar1, Amit Kumar-Dubey1, Nofar Glam-Matana1,2,AmirSherman4 and Dov Prusky1* Abstract Background: Penicillium expansum is a destructive phytopathogen that causes decay in deciduous fruits during postharvest handling and storage. During colonization the fungus secretes D-gluconic acid (GLA), which modulates environmental pH and regulates mycotoxin accumulation in colonized tissue. Till now no transcriptomic analysis has addressed the specific contribution of the pathogen's pH regulation to the P. expansum colonization process. For this purpose total RNA from the leading edge of P. expansum-colonized apple tissue of cv. 'Golden Delicious' and from fungal cultures grown under pH 4 or 7 were sequenced and their gene expression patterns were compared. Results: We present a large-scale analysis of the transcriptome data of P. expansum and apple response to fungal colonization. The fungal analysis revealed nine different clusters of gene expression patterns that were divided among three major groups in which the colonized tissue showed, respectively: (i) differing transcript expression patterns between mycelial growth at pH 4 and pH 7; (ii) similar transcript expression patterns of mycelial growth at pH 4; and (iii) similar transcript expression patterns of mycelial growth at pH 7. Each group was functionally characterized in order to decipher genes that are important for pH regulation and also for colonization of apple fruits by Penicillium. Furthermore, comparison of gene expression of healthy apple tissue with that of colonized tissue showed that differentially expressed genes revealed up-regulation of the jasmonic acid and mevalonate pathways, and also down-regulation of the glycogen and starch biosynthesis pathways. Conclusions: Overall, we identified important genes and functionalities of P. expansum that were controlled by the environmental pH. Differential expression patterns of genes belonging to the same gene family suggest that genes were selectively activated according to their optimal environmental conditions (pH, in vitro or in vivo) to enable the fungus to cope with varying conditions and to make optimal use of available enzymes. Comparison between the activation of the colonized host's gene responses by alkalizing Colletotrichum gloeosporioides and acidifying P. expansum pathogens indicated similar gene response patterns, but stronger responses to P. expansum, suggesting the importance of acidification by P. expansum as a factor in its increased aggressiveness. Keywords: Transcription profiling, Penicillium expansum,RNA-seq,Geneexpression,pHregulation,Pathogenicity, pH-regulated genes, Fungal genes regulated by pH, Apple genes regulated by pH * Correspondence: [email protected] †Equal contributors 1Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, the Volcani Center, Bet Dagan 50250, Israel Full list of author information is available at the end of the article © 2016 Barad et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Barad et al. BMC Genomics (2016) 17:330 Page 2 of 27 Background resident microorganisms [5] have suggested a wide var- The genus Penicillium comprises a group of anamorphic iety of genes that may contribute to the pathogenicity of fungi in the division Ascomycota [1]. Some Penicillium this pathogen. However, the work of Barad et al. [8] has species are of economic importance because they are attributed to the pH regulation of the host by P. expan- postharvest pathogens that cause spoilage in tropical and sum specific importance for pathogenicity. In the present deciduous fruits. Penicillium expansum is the causal agent study we used a transcriptomic approach to analyze the of blue mold, which is considered one of the most import- effect of pH on fungal gene regulation and host re- ant postharvest pathogens [2] and that causes decay in de- sponses to various pH-modulating pathogens. The effect ciduous fruits during postharvest handling and storage. of the fungal response was observed by comparing the Penicillium expansum causes extensive maceration of responses of the apple to colonization by P. expansum the infected tissue by means of a common mechanism as driven by expression of the fungal gene at pH 4 or 7. of tissue acidification [3]; it acidifies its host by activation Our RNA-Seq data of fungal responses revealed nine dif- of glucose oxidase 2 (gox2) and the catalyzed oxidation of ferentially co-expressed gene clusters; they showed alter- glucose resulting in secretion of small molecules such as ations of expression patterns that were associated with D-gluconic acid (GLA) [3, 4], which modulate the envir- differential pH responses, and also with genes related to onmental pH and thereby activate several polygalacturo- P. expansum colonization. The apple response to pH nases that contribute to pectin depolymerization and was elucidated by using qRT-PCR to analyze expressions consequently tissue maceration, at pH 3.5-4 [3, 5, 6]. of selected genes in response to acidifying and alkalinizing Functional analysis of glucose oxidase 2-RNAi mutants pathogens: P. expansum and Colletotrichum gloeospor- showed a strong effect on pathogen interactions with their ioides, respectively. This analysis indicated similar patterns host: the greater the down-regulation of gox2, the stronger of host response to the respective pathogens, but the apple the impairment of GLA production, medium acidification, genes showed an extensive multifold greater response to in- and apple fruit colonization [7]. More recently, Barad et al. fection by its natural pathogen P. expansum than to that by [8] reported that P. expansum secreted not only GLA but C. gloeosporioides. This may indicate the importance of the also ammonia at the leading edge of colonization in apple acidifying modulation of pH by Penicillium as a factor for fruits. Growth of the fungus in high-sucrose media induced greater aggressiveness of P. expansum. rapid metabolism of sugar and increased GLA accumula- tion, and the decrease in available carbon reserves resulted in enhanced ammonia accumulation, probably because of Results and Discussion amino acid catabolism, such as occurs with other pathogens Profiling the expression of P. expansum genes during [9, 10]. These results indicated that bidirectional pH colonization and growth at pH 4 and 7 regulation occurs in P. expansum-probably dependent Genomes of P. expansum [7] were sequenced by using on nutrient availability. The question concerns which paired-end reads by means of Illumina Hiseq 2500 [12]. For metabolic processes are modulated by the pH level dur- analysis of the P. expansum transcriptomewedownloaded ing colonization of apple fruits. the reference draft genome accession JQFX00000000.1 PacC is a transcription factor that activates genes of from the Genbank site [13]. Ten libraries of single-end Aspergillus nidulans during the alkalization of the envir- RNAseq (deposited in Genbank under accession SRP071104) onment [11]. In a recent report [8], it was suggested that were mapped to the reference genome by using the P. expansum's pacC gene was active also under acidic Bowtie2 software [14]. The 10 libraries contained the conditions, and that ammonia produced at the leading following features: (i) P. expansum grown in culture media edge of the decaying tissue under low-pH conditions fur- at pH 4 for 3 h as well as a pooled sample collected at sev- ther contributed to the activation of pacC responsiveness. eral time points-0.5, 1, 3, 10, and 24 h-with two replicates This may indicate that P. expansum uses ammonia accu- at each point; (ii) P. expansum grown in culture media at mulation during nutritional modification of the ambient pH 7 for 3 h, as well as a pool collected at several time environmental pH as a regulatory cue for activation of points-0.5, 1, 3, 10, and 24 h-with two replicates at each pacC, by signaling and activating alkaline-induced genes point; and (iii) the leading edge, comprising 1–2mmof that contribute to pathogenicity and accumulation of sec- apple cv. 'Golden Delicious' tissue, colonized by P. ondary metabolites such as patulin [8]. This pH modulation expansum. For in vitro experiments, 106 spores of P. under the influence of GLA and ammonia accumulation is expansum were inoculated into germinating minimal important for understanding the fungal response to differ- medium (GMM) at pH 4.5 as described by Barad et al. ential gene regulation by pH; however, the overall response [7] and transferred 2 days later to buffered inducing remains poorly understood. media at pH 4 or 7. At several different time points after The diversity of recently described ecological interac- transfer to inducing secondary media (SM), fungal mycelia tions of Penicillium with fruits [3, 4] and with other were sampled and pooled for analysis. Pooled samples of Barad et al. BMC Genomics (2016) 17:330 Page 3 of 27 RNA were compared with independent samples 3 h after Group I comprised clusters of genes whose expression transfer to inducing media. patterns in the colonized tissue differed with respect to Principal component analysis (PCA) of all expressed growth at pH 4 or pH 7. This group comprised clusters genes of P. expansum indicated that the replicates were 1, 2, 3, and 6, which contained genes that are important very close to each other, and that the expression patterns for P. expansum colonization within apple fruits.
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